Evaluation of a novel mitochondrial Pan-Mucorales marker for the detection, identification, quantification, and growth stage determination of mucormycetes

Mucormycosis infections are infrequent yet aggressive and serious fungal infections. Early diagnosis of mucormycosis and its discrimination from other fungal infections is required for targeted treatment and more favorable patient outcomes. The majority of the molecular assays use 18 S rDNA. In the current study, we aimed to explore the potential of the mitochondrial rnl (encoding for large-subunit-ribosomal-RNA) gene as a novel molecular marker suitable for research and diagnostics. Rnl was evaluated as a marker for: (1) the Mucorales family, (2) species identification (Rhizopus arrhizus, R. microsporus, Mucor circinelloides, and Lichtheimia species complexes), (3) growth stage, and (4) quantification. Sensitivity, specificity, discriminatory power, the limit of detection (LoD), and cross-reactivity were evaluated. Assays were tested using pure cultures, spiked clinical samples, murine organs, and human paraffin-embedded-tissue (FFPE) samples. Mitochondrial markers were found to be superior to nuclear markers for degraded samples. Rnl outperformed the UMD universal® (Molyzm) marker in FFPE (71.5% positive samples versus 50%). Spiked blood samples highlighted the potential of rnl as a pan-Mucorales screening test. Fungal burden was reproducibly quantified in murine organs using standard curves. Identification of pure cultures gave a perfect (100%) correlation with the detected internal transcribed spacer (ITS) sequence. In conclusion, mitochondrial genes, such as rnl, provide an alternative to the nuclear 18 S rDNA genes and deserve further evaluation.CD laboratory: This research was funded by the Christian Doppler Laboratory for fungal infections

Predictive value of neurological examination for early cortical responses to somatosensory evoked potentials in patients with postanoxic coma

Bilateral absence of cortical N20 responses of median nerve somatosensory evoked potentials (SEP) predicts poor neurological outcome in postanoxic coma after cardiopulmonary resuscitation (CPR). Although SEP is easy to perform and available in most hospitals, it is worthwhile to know how neurological signs are associated with SEP results. The aim of this study was to investigate whether specific clinical neurological signs are associated with either an absent or a present median nerve SEP in patients after CPR. Data from the previously published multicenter prospective cohort study PROPAC (prognosis in postanoxic coma, 2000–2003) were used. Neurological examination, consisting of Glasgow Coma Score (GCS) and brain stem reflexes, and SEP were performed 24, 48, and 72 h after CPR. Positive predictive values for predicting absent and present SEP, as well as diagnostic accuracy were calculated. Data of 407 patients were included. Of the 781 SEPs performed, N20 s were present in 401, bilaterally absent in 299, and 81 SEPs were technically undeterminable. The highest positive predictive values (0.63–0.91) for an absent SEP were found for absent pupillary light responses. The highest positive predictive values (0.71–0.83) for a present SEP were found for motor scores of withdrawal to painful stimuli or better. Multivariate analyses showed a fair diagnostic accuracy (0.78) for neurological examination in predicting an absent or present SEP at 48 or 72 h after CPR. This study shows that neurological examination cannot reliably predict absent or present cortical N20 responses in median nerve SEPs in patients after CPR

Phytochrome-Based Extracellular Matrix with Reversibly Tunable mechanical Properties

Interrogation and control of cellular fate and function using optogenetics is providing revolutionary insights into biology. Optogenetic control of cells is achieved by coupling genetically encoded photoreceptors to cellular effectors and enables unprecedented spatiotemporal control of signaling processes. Here, a fast and reversibly switchable photoreceptor is used to tune the mechanical properties of polymer materials in a fully reversible, wavelength‐specific, and dose‐ and space‐controlled manner. By integrating engineered cyanobacterial phytochrome 1 into a poly(ethylene glycol) matrix, hydrogel materials responsive to light in the cell‐compatible red/far‐red spectrum are synthesized. These materials are applied to study in human mesenchymal stem cells how different mechanosignaling pathways respond to changing mechanical environments and to control the migration of primary immune cells in 3D. This optogenetics‐inspired matrix allows fundamental questions of how cells react to dynamic mechanical environments to be addressed. Further, remote control of such matrices can create new opportunities for tissue engineering or provide a basis for optically stimulated drug depots

Multiple regulatory intrinsically disordered motifs control FOXO4 transcription factor binding and function

Transcription factors harbor defined regulatory intrinsically disordered regions (IDRs), which raises the question of how they mediate binding to structured co-regulators and modulate their activity. Here, we present a detailed molecular regulatory mechanism of Forkhead box O4 (FOXO4) by the structured transcriptional co-regulator β-catenin. We find that the disordered FOXO4 C-terminal region, which contains its transactivation domain, binds β-catenin through two defined interaction sites, and this is regulated by combined PKB/AKT- and CK1-mediated phosphorylation. Binding of β-catenin competes with the autoinhibitory interaction of the FOXO4 disordered region with its DNA-binding Forkhead domain, and thereby enhances FOXO4 transcriptional activity. Furthermore, we show that binding of the β-catenin inhibitor protein ICAT is compatible with FOXO4 binding to β-catenin, suggesting that ICAT acts as a molecular switch between anti-proliferative FOXO and pro-proliferative Wnt/TCF/LEF signaling. These data illustrate how the interplay of IDRs, post-translational modifications, and co-factor binding contribute to transcription factor function

Design-time formal verification for smart environments: an exploratory perspective

Smart environments (SmE) are richly integrated with multiple heterogeneous devices; they perform the operations in intelligent manner by considering the context and actions/behaviors of the users. Their major objective is to enable the environment to provide ease and comfort to the users. The reliance on these systems demands consistent behavior. The versatility of devices, user behavior and intricacy of communication complicate the modeling and verification of SmE's reliable behavior. Of the many available modeling and verification techniques, formal methods appear to be the most promising. Due to a large variety of implementation scenarios and support for conditional behavior/processing, the concept of SmE is applicable to diverse areas which calls for focused research. As a result, a number of modeling and verification techniques have been made available for designers. This paper explores and puts into perspective the modeling and verification techniques based on an extended literature survey. These techniques mainly focus on some specific aspects, with a few overlapping scenarios (such as user interaction, devices interaction and control, context awareness, etc.), which were of the interest to the researchers based on their specialized competencies. The techniques are categorized on the basis of various factors and formalisms considered for the modeling and verification and later analyzed. The results show that no surveyed technique maintains a holistic perspective; each technique is used for the modeling and verification of specific SmE aspects. The results further help the designers select appropriate modeling and verification techniques under given requirements and stress for more R&D effort into SmE modeling and verification researc

A Helix Replacement Mechanism Directs Metavinculin Functions

Cells require distinct adhesion complexes to form contacts with their neighbors or the extracellular matrix, and vinculin links these complexes to the actin cytoskeleton. Metavinculin, an isoform of vinculin that harbors a unique 68-residue insert in its tail domain, has distinct actin bundling and oligomerization properties and plays essential roles in muscle development and homeostasis. Moreover, patients with sporadic or familial mutations in the metavinculin-specific insert invariably develop fatal cardiomyopathies. Here we report the high resolution crystal structure of the metavinculin tail domain, as well as the crystal structures of full-length human native metavinculin (1,134 residues) and of the full-length cardiomyopathy-associated ΔLeu954 metavinculin deletion mutant. These structures reveal that an α-helix (H1′) and extended coil of the metavinculin insert replace α-helix H1 and its preceding extended coil found in the N-terminal region of the vinculin tail domain to form a new five-helix bundle tail domain. Further, biochemical analyses demonstrate that this helix replacement directs the distinct actin bundling and oligomerization properties of metavinculin. Finally, the cardiomyopathy associated ΔLeu954 and Arg975Trp metavinculin mutants reside on the replaced extended coil and the H1′ α-helix, respectively. Thus, a helix replacement mechanism directs metavinculin's unique functions

Polymerization-Induced Self-Assembly of Block Copolymer Nano-objects via RAFT Aqueous Dispersion Polymerization

In this Perspective, we discuss the recent development of polymerization-induced self-assembly mediated by reversible addition–fragmentation chain transfer (RAFT) aqueous dispersion polymerization. This approach has quickly become a powerful and versatile technique for the synthesis of a wide range of bespoke organic diblock copolymer nano-objects of controllable size, morphology, and surface functionality. Given its potential scalability, such environmentally-friendly formulations are expected to offer many potential applications, such as novel Pickering emulsifiers, efficient microencapsulation vehicles, and sterilizable thermo-responsive hydrogels for the cost-effective long-term storage of mammalian cells

A target-protection mechanism of antibiotic resistance at atomic resolution: insights into FusB-type fusidic acid resistance

Antibiotic resistance in clinically important bacteria can be mediated by proteins that physically associate with the drug target and act to protect it from the inhibitory effects of an antibiotic. We present here the first detailed structural characterization of such a target protection mechanism mediated through a protein-protein interaction, revealing the architecture of the complex formed between the FusB fusidic acid resistance protein and the drug target (EF-G) it acts to protect. Binding of FusB to EF G induces conformational and dynamic changes in the latter, shedding light on the molecular mechanism of fusidic acid resistance